Engineered enzymes

ABSTRACT

The present disclosure provides engineered RNA-guided enzymes for editing live cells.

RELATED APPLICATIONS

This application is a continuation of U.S. Ser. No. 16/844,079, filed 9Apr. 2020, now allowed; which is a continuation of U.S. Ser. No.16/798,315, filed 22 Feb. 2020, now U.S. Pat. No. 10,640,754; which is acontinuation of U.S. Ser. No. 16/658,948, filed 21 Oct. 2019, now U.S.Pat. No. 10,604,746; which claims priority to U.S. ProvisionalApplication No. 62/748,668, filed 22 Oct. 2018.

FIELD OF THE INVENTION

This invention relates to engineered enzymes for editing live cells.

BACKGROUND OF THE INVENTION

In the following discussion certain articles and methods will bedescribed for background and introductory purposes. Nothing containedherein is to be construed as an “admission” of prior art. Applicantexpressly reserves the right to demonstrate, where appropriate, that themethods referenced herein do not constitute prior art under theapplicable statutory provisions.

The ability to make precise, targeted changes to the genome of livingcells has been a long-standing goal in biomedical research anddevelopment. Recently, various nucleases have been identified that allowmanipulation of gene sequence, and hence gene function. These nucleasesinclude nucleic acid-guided nucleases. The range of target sequencesthat nucleic acid-guided nucleases can recognize, however, isconstrained by the need for a specific protospacer adjacent motif (PAM)to be located near the desired target sequence. PAMs are shortnucleotide sequences recognized by a gRNA/nuclease complex, where thiscomplex directs editing of a target sequence in a live cell. The precisePAM sequence and length requirements for different nucleic acid-guidednucleases vary; however, PAMs typically are 2-7 base-pair sequencesadjacent or in proximity to the target sequence and, depending on thenuclease, can be 5′ or 3′ to the target sequence. Engineering of nucleicacid-guided nucleases may allow for alteration of PAM preference, allowfor editing optimization in different organisms and/or alter enzymefidelity; all changes that may increase the versatility of a specificnucleic acid-guided nuclease for certain editing tasks.

There is thus a need in the art of nucleic acid-guided nuclease geneediting for improved nucleases. The engineered MAD70-series nucleasesdescribed herein satisfy this need.

SUMMARY OF THE INVENTION

This Summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key or essentialfeatures of the claimed subject matter, nor is it intended to be used tolimit the scope of the claimed subject matter. Other features, details,utilities, and advantages of the claimed subject matter will be apparentfrom the following written Detailed Description including those aspectsillustrated in the accompanying drawings and defined in the appendedclaims.

The present disclosure provides engineered MAD70-series nucleases withvaried PAM preferences, varied editing efficiency in different organismsand/or altered RNA-guided enzyme fidelity (e.g., decreased off-targetcutting).

Thus, in one embodiment there is provided an engineered MAD70-seriesnuclease with an altered RNA-guided enzyme fidelity relative to the MAD7nuclease where the MAD7 nuclease has the amino acid sequence of SEQ IDNo. 1. In some aspects of this embodiment, the engineered MAD70-seriesnuclease with the higher altered RNA-guided enzyme fidelity comprisesany of SEQ ID No. 4-7.

In other embodiments there is provided an engineered MAD70-seriesnuclease having a PAM preference different than the MAD7 nuclease havingthe sequence of SEQ ID No. 1. In some aspects of this embodiment, theengineered MAD70-series nuclease having an altered PAM preferencecomprises any of SEQ ID Nos. 2, 3, 11, 12, 13, 14, 67 or 68. In someaspects of this embodiment, there is provided a cocktail of nucleaseenzymes comprising one, two, three, four, five or all of SEQ ID Nos. 2,3, 11, 12, 13, 14, 67 or 68, and in some aspects, there is provided acocktail of nuclease enzymes comprising one, some or all of SEQ ID Nos.2, 3, 11, 12, 13, 14, 67 or 68 and another nuclease with a PAMpreference different from SEQ ID Nos. 2, 3, 11, 12, 13, 14, 67 or 68,and in some aspects, the other nuclease has a sequence of SEQ ID No. 4,5, 6, 7, 69-78 or 79-86.

Additionally, there is provided is an engineered MAD70-series nucleasewith lower cutting activity relative to the MAD7 nuclease having thesequence of SEQ ID No. 1. In some aspects of this embodiment, theengineered MAD70-series nuclease having lowered cutting activitycomprises any of SEQ ID Nos. 8-10 or 15.

Also there is provided an engineered MAD70-series nuclease with enhancedediting efficiency in yeast comprising any of SEQ ID Nos. 69-78 and79-86.

These aspects and other features and advantages of the invention aredescribed below in more detail.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a heatmap for certain of the MAD70-series nucleases withdifferent PAM recognition sites. FIG. 1B-1 is a heatmap for certain ofthe MAD70-series nucleases with varied fidelity as compared with theMAD7 (SEQ ID No.1). FIG. B-2 shows the PAM depletion panel for the sameenzyme from FIG. B-1.

FIGS. 2A and 2B show the results of 108 engineered MAD70-seriesnucleases selected from screening 1104 single amino acid variants. FIG.2A is the plot (sum of PAM depletion vs. pos9_score) for the MAD7nuclease having the sequence SEQ ID NO. 1, and FIG. 2B is the plot forthe screened 1104 single amino acid variants.

FIG. 3 is an exemplary workflow for creating and screening engineeredMAD70-series enzymes.

FIG. 4 shows the sequence of two different gRNA constructs used fordepletion studies (SEQ ID No. 21-24).

FIG. 5 is a heatmap for PAM preferences for MAD70-series variants from acombinatorial library screen.

FIG. 6A is a complete NNNN PAM preference for wild-type MAD 7 (SEQ IDNo. 1). FIG. 6B is a complete NNNN PAM preference for the K535R/N539Smutant (SEQ ID No. 67). FIG. 6C is a complete NNNN PAM preference forthe K535R/N539S/K594L/E730Q mutant (SEQ ID No. 68).

FIG. 7 shows colonies containing editing cassettes and wild-type MAD7,MAD70-series variants K535R/N539S (SEQ ID No. 67) andK535R/N539S/K594L/E730Q (SEQ ID No. 68) mutants in relation to thewild-type MAD7 amino acid sequence.

FIG. 8 is a map of the plasmid used for the screening of nucleaseproteins for genome editing activity in S. cerevisiae.

FIG. 9 shows the relative rates of genome editing at different positionsof the Can1 protein locus with the indicated PAM by wild-type MAD7, andthe K535R (SEQ ID No. 13), N539A and K535R/N539S (SEQ ID No. 67)MAD70-series mutants.

FIG. 10 shows the results of screening 2304 MAD70-series variants forgenome editing activity in S. cerevisiae.

FIG. 11 shows quadruplicate re-testing of MAD70-series variants thatdemonstrated enhanced genome editing activity in S. cerevisiae.

FIG. 12 shows the results of screening 2304 MAD70-series combinatorialprotein variants for genome editing activity in S. cerevisiae.

FIG. 13 shows the results of secondary screening of the MAD70-seriescombinatorial variant hits showing fractional difference in genomeediting activity in S. cerevisiae and the multiple-comparison-adjusted Pvalue for each variant as compared to the wild-type MAD 7 (SEQ ID No. 1)controls.

FIG. 14 shows the results of genome editing in mammalian HEK293T cellswith wild-type MAD7 (SEQ ID No. 1) and MAD70-series variants withAsCas12a as a control.

DETAILED DESCRIPTION

The description set forth below in connection with the appended drawingsis intended to be a description of various, illustrative embodiments ofthe disclosed subject matter. Specific features and functionalities aredescribed in connection with each illustrative embodiment; however, itwill be apparent to those skilled in the art that the disclosedembodiments may be practiced without each of those specific features andfunctionalities. Moreover, all of the functionalities described inconnection with one embodiment are intended to be applicable to theadditional embodiments described herein except where expressly stated orwhere the feature or function is incompatible with the additionalembodiments. For example, where a given feature or function is expresslydescribed in connection with one embodiment but not expressly mentionedin connection with an alternative embodiment, it should be understoodthat the feature or function may be deployed, utilized, or implementedin connection with the alternative embodiment unless the feature orfunction is incompatible with the alternative embodiment.

The practice of the techniques described herein may employ, unlessotherwise indicated, conventional techniques and descriptions of organicchemistry, polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry, biological emulsion generation,and sequencing technology, which are within the skill of those whopractice in the art. Such conventional techniques include polymer arraysynthesis, hybridization and ligation of polynucleotides, and detectionof hybridization using a label. Specific illustrations of suitabletechniques can be had by reference to the examples herein. However,other equivalent conventional procedures can, of course, also be used.Such conventional techniques and descriptions can be found in standardlaboratory manuals such as Green, et al., Eds. (1999), Genome Analysis:A Laboratory Manual Series (Vols. I-IV); Weiner, Gabriel, Stephens, Eds.(2007), Genetic Variation: A Laboratory Manual; Dieffenbach, Dveksler,Eds. (2003), PCR Primer: A Laboratory Manual; Bowtell and Sambrook(2003), DNA Microarrays: A Molecular Cloning Manual; Mount (2004),Bioinformatics: Sequence and Genome Analysis; Sambrook and Russell(2006), Condensed Protocols from Molecular Cloning: A Laboratory Manual;and Sambrook and Russell (2002), Molecular Cloning: A Laboratory Manual(all from Cold Spring Harbor Laboratory Press); Stryer, L. (1995)Biochemistry (4th Ed.) W.H. Freeman, New York N.Y.; Gait,“Oligonucleotide Synthesis: A Practical Approach” 1984, IRL Press,London; Nelson and Cox (2000), Lehninger, Principles of Biochemistry3^(rd) Ed., W. H. Freeman Pub., New York, N.Y.; Berg et al. (2002)Biochemistry, 5^(th) Ed., W.H. Freeman Pub., New York, N.Y.; Cell andTissue Culture: Laboratory Procedures in Biotechnology (Doyle &Griffiths, eds., John Wiley & Sons 1998); Mammalian ChromosomeEngineering—Methods and Protocols (G. Hadlaczky, ed., Humana Press2011); Essential Stem Cell Methods, (Lanza and Klimanskaya, eds.,Academic Press 2011), all of which are herein incorporated in theirentirety by reference for all purposes. Nuclease-specific techniques canbe found in, e.g., Genome Editing and Engineering From TALENs andCRISPRs to Molecular Surgery, Appasani and Church, 2018; and CRISPR:Methods and Protocols, Lindgren and Charpentier, 2015; both of which areherein incorporated in their entirety by reference for all purposes.Basic methods for enzyme engineering may be found in, Enzyme EngineeringMethods and Protocols, Samuelson, ed., 2013; Protein Engineering,Kaumaya, ed., (2012); and Kaur and Sharma, “Directed Evolution: AnApproach to Engineer Enzymes”, Crit. Rev. Biotechnology, 26:165-69(2006).

Note that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “an oligonucleotide”refers to one or more oligonucleotides, and reference to “an automatedsystem” includes reference to equivalent steps and methods for use withthe system known to those skilled in the art, and so forth.Additionally, it is to be understood that terms such as “left,” “right,”“top,” “bottom,” “front,” “rear,” “side,” “height,” “length,” “width,”“upper,” “lower,” “interior,” “exterior,” “inner,” “outer” that may beused herein merely describe points of reference and do not necessarilylimit embodiments of the present disclosure to any particularorientation or configuration. Furthermore, terms such as “first,”“second,” “third,” etc., merely identify one of a number of portions,components, steps, operations, functions, and/or points of reference asdisclosed herein, and likewise do not necessarily limit embodiments ofthe present disclosure to any particular configuration or orientation.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications mentionedherein are incorporated by reference for the purpose of describing anddisclosing devices, methods and cell populations that may be used inconnection with the presently described invention.

Where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the invention. The upper and lower limits of thesesmaller ranges may independently be included in the smaller ranges, andare also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either both of those includedlimits are also included in the invention.

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present invention. However,it will be apparent to one of ordinary skill in the art that the presentinvention may be practiced without one or more of these specificdetails. In other instances, well-known features and procedures wellknown to those skilled in the art have not been described in order toavoid obscuring the invention.

The term “complementary” as used herein refers to Watson-Crick basepairing between nucleotides and specifically refers to nucleotideshydrogen bonded to one another with thymine or uracil residues linked toadenine residues by two hydrogen bonds and cytosine and guanine residueslinked by three hydrogen bonds. In general, a nucleic acid includes anucleotide sequence described as having a “percent complementarity” or“percent homology” to a specified second nucleotide sequence. Forexample, a nucleotide sequence may have 80%, 90%, or 100%complementarity to a specified second nucleotide sequence, indicatingthat 8 of 10, 9 of 10 or 10 of 10 nucleotides of a sequence arecomplementary to the specified second nucleotide sequence. For instance,the nucleotide sequence 3′-TCGA-5′ is 100% complementary to thenucleotide sequence 5′-AGCT-3′; and the nucleotide sequence 3′-TCGA-5′is 100% complementary to a region of the nucleotide sequence5′-TTAGCTGG-3′.

The term DNA “control sequences” refers collectively to promotersequences, polyadenylation signals, transcription termination sequences,upstream regulatory domains, origins of replication, internal ribosomeentry sites, nuclear localization sequences, enhancers, and the like,which collectively provide for the replication, transcription andtranslation of a coding sequence in a recipient cell. Not all of thesetypes of control sequences need to be present so long as a selectedcoding sequence is capable of being replicated, transcribed and—for somecomponents—translated in an appropriate host cell.

As used herein the term “donor DNA” or “donor nucleic acid” refers tonucleic acid that is designed to introduce a DNA sequence modification(insertion, deletion, substitution) into a locus by homologousrecombination using nucleic acid-guided nucleases. For homology-directedrepair, the donor DNA must have sufficient homology to the regionsflanking the “cut site” or site to be edited in the genomic targetsequence. The length of the homology arm(s) will depend on, e.g., thetype and size of the modification being made. In many instances andpreferably, the donor DNA will have two regions of sequence homology(e.g., two homology arms) to the genomic target locus. Preferably, an“insert” region or “DNA sequence modification” region—the nucleic acidmodification that one desires to be introduced into a genome targetlocus in a cell-will be located between two regions of homology. The DNAsequence modification may change one or more bases of the target genomicDNA sequence at one specific site or multiple specific sites. A changemay include changing 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 50, 75,100, 150, 200, 300, 400, or 500 or more base pairs of the targetsequence. A deletion or insertion may be a deletion or insertion of 1,2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, 75, 100, 150, 200, 300, 400, or500 or more base pairs of the target sequence.

The terms “guide nucleic acid” or “guide RNA” or “gRNA” refer to apolynucleotide comprising 1) a guide sequence capable of hybridizing toa genomic target locus, and 2) a scaffold sequence capable ofinteracting or complexing with a nucleic acid-guided nuclease.

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or, more often in the context of the presentdisclosure, between two nucleic acid molecules. The term “homologousregion” or “homology arm” refers to a region on the donor DNA with acertain degree of homology with the target genomic DNA sequence.Homology can be determined by comparing a position in each sequencewhich may be aligned for purposes of comparison. When a position in thecompared sequence is occupied by the same base or amino acid, then themolecules are homologous at that position. A degree of homology betweensequences is a function of the number of matching or homologouspositions shared by the sequences.

“Operably linked” refers to an arrangement of elements where thecomponents so described are configured so as to perform their usualfunction. Thus, control sequences operably linked to a coding sequenceare capable of effecting the transcription, and in some cases, thetranslation, of a coding sequence. The control sequences need not becontiguous with the coding sequence so long as they function to directthe expression of the coding sequence. Thus, for example, interveninguntranslated yet transcribed sequences can be present between a promotersequence and the coding sequence and the promoter sequence can still beconsidered “operably linked” to the coding sequence. In fact, suchsequences need not reside on the same contiguous DNA molecule (i.e.chromosome) and may still have interactions resulting in alteredregulation.

A “promoter” or “promoter sequence” is a DNA regulatory region capableof binding RNA polymerase and initiating transcription of apolynucleotide or polypeptide coding sequence such as messenger RNA,ribosomal RNA, small nuclear or nucleolar RNA, guide RNA, or any kind ofRNA transcribed by any class of any RNA polymerase I, II or III.Promoters may be constitutive or inducible and, in someembodiments—particularly many embodiments in which selection isemployed—the transcription of at least one component of the nucleicacid-guided nuclease editing system is under the control of an induciblepromoter.

As used herein the term “selectable marker” refers to a gene introducedinto a cell, which confers a trait suitable for artificial selection.General use selectable markers are well-known to those of ordinary skillin the art. Drug selectable markers such as ampicillin/carbenicillin,kanamycin, chloramphenicol, erythromycin, tetracycline, gentamicin,bleomycin, streptomycin, rhamnose, puromycin, hygromycin, blasticidin,and G418 may be employed. In other embodiments, selectable markersinclude, but are not limited to human nerve growth factor receptor(detected with a MAb, such as described in U.S. Pat. No. 6,365,373);truncated human growth factor receptor (detected with MAb); mutant humandihydrofolate reductase (DHFR; fluorescent MTX substrate available);secreted alkaline phosphatase (SEAP; fluorescent substrate available);human thymidylate synthase (TS; confers resistance to anti-cancer agentfluorodeoxyuridine); human glutathione S-transferase alpha (GSTA1;conjugates glutathione to the stem cell selective alkylator busulfan;chemoprotective selectable marker in CD34+ cells); CD24 cell surfaceantigen in hematopoietic stem cells; human CAD gene to confer resistanceto N-phosphonacetyl-L-aspartate (PALA); human multi-drug resistance-1(MDR-1; P-glycoprotein surface protein selectable by increased drugresistance or enriched by FACS); human CD25 (IL-2a; detectable byMab-FITC); Methylguanine-DNA methyltransferase (MGMT; selectable bycarmustine); and Cytidine deaminase (CD; selectable by Ara-C).“Selective medium” as used herein refers to cell growth medium to whichhas been added a chemical compound or biological moiety that selects foror against selectable markers.

The terms “target genomic DNA sequence”, “target sequence”, or “genomictarget locus” refer to any locus in vitro or in vivo, or in a nucleicacid (e.g., genome) of a cell or population of cells, in which a changeof at least one nucleotide is desired using a nucleic acid-guidednuclease editing system. The target sequence can be a genomic locus orextrachromosomal locus.

A “vector” is any of a variety of nucleic acids that comprise a desiredsequence or sequences to be delivered to and/or expressed in a cell.Vectors are typically composed of DNA, although RNA vectors are alsoavailable. Vectors include, but are not limited to, plasmids, fosmids,phagemids, virus genomes, synthetic chromosomes, and the like. As usedherein, the phrase “engine vector” comprises a coding sequence for anuclease to be used in the nucleic acid-guided nuclease systems andmethods of the present disclosure. The engine vector may also comprise,in a bacterial system, the λ Red recombineering system or an equivalentthereto. Engine vectors also typically comprise a selectable marker. Asused herein the phrase “editing vector” comprises a donor nucleic acid,optionally including an alteration to the target sequence that preventsnuclease binding at a PAM or spacer in the target sequence after editinghas taken place, and a coding sequence for a gRNA. The editing vectormay also comprise a selectable marker and/or a barcode. In someembodiments, the engine vector and editing vector may be combined; thatis, the contents of the engine vector may be found on the editingvector. Further, the engine and editing vectors comprise controlsequences operably linked to, e.g., the nuclease coding sequence,recombineering system coding sequences (if present), donor nucleic acid,guide nucleic acid, and selectable marker(s).

Editing in Nucleic Acid-Guided Nuclease Genome Systems Generally

The present disclosure provides engineered gene editing MAD70-seriesnucleases with varied PAM preferences, optimized editing efficiency indifferent organisms, and/or an altered RNA-guided enzyme fidelity. Theengineered MAD70-series nucleases may be used to edit all cell typesincluding, archaeal, prokaryotic, and eukaryotic (e.g., yeast, fungal,plant and animal) cells although certain MAD70-series variants exhibitenhanced efficiency in, e.g., yeast or mammalian cells.

The engineered MAD70-series nuclease variants described herein improveRNA-guided enzyme editing systems in which nucleic acid-guided nucleases(e.g., RNA-guided nucleases) are used to edit specific target regions inan organism's genome. A nucleic acid-guided nuclease complexed with anappropriate synthetic guide nucleic acid in a cell can cut the genome ofthe cell at a desired location. The guide nucleic acid helps the nucleicacid-guided nuclease recognize and cut the DNA at a specific targetsequence. By manipulating the nucleotide sequence of the guide nucleicacid, the nucleic acid-guided nuclease may be programmed to target anyDNA sequence for cleavage as long as an appropriate protospacer adjacentmotif (PAM) is nearby.

The engineered MAD70-series nucleases may be delivered to cells to beedited as a polypeptide; alternatively, a polynucleotide sequenceencoding the engineered MAD70-series nuclease(s) is transformed ortransfected into the cells to be edited. The polynucleotide sequenceencoding the engineered MAD70-series nuclease may be codon optimized forexpression in particular cells, such as archaeal, prokaryotic oreukaryotic cells. Eukaryotic cells can be yeast, fungi, algae, plant,animal, or human cells. Eukaryotic cells may be those of or derived froma particular organism, such as a mammal, including but not limited tohuman, mouse, rat, rabbit, dog, or non-human mammals including non-humanprimates. The choice of the engineered MAD70-series nuclease to beemployed depends on many factors, such as what type of edit is to bemade in the target sequence and whether an appropriate PAM is locatedclose to the desired target sequence. The engineered MAD70-seriesnuclease may be encoded by a DNA sequence on a vector (e.g., the enginevector) and be under the control of a constitutive or induciblepromoter. In some embodiments, the sequence encoding the nuclease isunder the control of an inducible promoter, and the inducible promotermay be separate from but the same as an inducible promoter controllingtranscription of the guide nucleic acid; that is, a separate induciblepromoter may drive the transcription of the nuclease and guide nucleicacid sequences but the two inducible promoters may be the same type ofinducible promoter. Alternatively, the inducible promoter controllingexpression of the nuclease may be different from the inducible promotercontrolling transcription of the guide nucleic acid.

In general, a guide nucleic acid (e.g., gRNA) complexes with acompatible nucleic acid-guided nuclease and can then hybridize with atarget sequence, thereby directing the nuclease to the target sequence.In certain aspects, the RNA-guided enzyme editing system may use twoseparate guide nucleic acid molecules that combine to function as aguide nucleic acid, e.g., a CRISPR RNA (crRNA) and trans-activatingCRISPR RNA (tracrRNA). In other aspects—and used with the MAD70-seriesvariant nucleases described herein—the guide nucleic acid may be asingle guide nucleic acid that includes both the crRNA and tracrRNAsequences. A guide nucleic acid can be DNA or RNA; alternatively, aguide nucleic acid may comprise both DNA and RNA. In some embodiments, aguide nucleic acid may comprise modified or non-naturally occurringnucleotides. In cases where the guide nucleic acid comprises RNA, thegRNA may be encoded by a DNA sequence on a polynucleotide molecule suchas a plasmid, linear construct, or the coding sequence may reside withinan editing cassette and is under the control of a constitutive promoter,or, in some embodiments, an inducible promoter as described below.

A guide nucleic acid comprises a guide sequence, where the guidesequence is a polynucleotide sequence having sufficient complementaritywith a target sequence to hybridize with the target sequence and directsequence-specific binding of a complexed nucleic acid-guided nuclease tothe target sequence. The degree of complementarity between a guidesequence and the corresponding target sequence, when optimally alignedusing a suitable alignment algorithm, is about or more than about 50%,60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment maybe determined with the use of any suitable algorithm for aligningsequences. In some embodiments, a guide sequence is about or more thanabout 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length.In some embodiments, a guide sequence is less than about 75, 50, 45, 40,35, 30, 25, 20 nucleotides in length. Preferably the guide sequence is10-30 or 15-20 nucleotides long, or 15, 16, 17, 18, 19, or 20nucleotides in length.

In the present methods and compositions, the guide nucleic acidtypically is provided as a sequence to be expressed from a plasmid orvector and comprises both the guide sequence and the scaffold sequenceas a single transcript under the control of a promoter, and in someembodiments, an inducible promoter. The guide nucleic acid can beengineered to target a desired target sequence by altering the guidesequence so that the guide sequence is complementary to a desired targetsequence, thereby allowing hybridization between the guide sequence andthe target sequence. In general, to generate an edit in the targetsequence, the gRNA/nuclease complex binds to a target sequence asdetermined by the guide RNA, and the nuclease recognizes a photospaceradjacent motif TAM) sequence adjacent to the target sequence. The targetsequence can be any polynucleotide endogenous or exogenous to aprokaryotic or eukaryotic cell, or in vitro. For example, the targetsequence can be a polynucleotide residing in the nucleus of a eukaryoticcell. A target sequence can be a sequence encoding a gene product (e.g.,a protein) or a non-coding sequence (e.g., a regulatory polynucleotide,an intron, a PAM, or “junk” DNA).

The guide nucleic acid may be part of an editing cassette that encodesthe donor nucleic acid, such as described in U.S. Pat. No. 10,240,167,issued 26 Mar. 2019; U.S. Pat. No. 10,266,849, issued 23 Apr. 2019; U.S.Pat. No. 9,982,278, issued 22 Jun. 2018; U.S. Pat. No. 10,351,877,issued 15 Jul. 2019; and U.S. Pat. No. 10,362,422, issued 30 Jul. 2019;and U.S. Ser. No. 16/275,439, filed 14 Feb. 2019; Ser. No. 16/275,465,filed 14 Feb. 2019; Ser. No. 16/550,092, filed 23 Aug. 2019; and Ser.No. 16/552,517, filed 26 Aug. 2019. Alternatively, the guide nucleicacid may not be part of the editing cassette and instead may be encodedon the engine or editing vector backbone. For example, a sequence codingfor a guide nucleic acid can be assembled or inserted into a vectorbackbone first, followed by insertion of the donor nucleic acid in,e.g., the editing cassette. In other cases, the donor nucleic acid in,e.g., an editing cassette can be inserted or assembled into a vectorbackbone first, followed by insertion of the sequence coding for theguide nucleic acid. In yet other cases, the sequence encoding the guidenucleic acid and the donor nucleic acid (inserted, for example, in anediting cassette) are simultaneously but separately inserted orassembled into a vector. In yet other embodiments, the sequence encodingthe guide nucleic acid and the sequence encoding the donor nucleic acidare both included in the editing cassette.

The target sequence is associated with a PAM, which is a shortnucleotide sequence recognized by the gRNA/nuclease complex. The precisePAM sequence and length requirements for different nucleic acid-guidednucleases vary; however, PAMs typically are 2-7 base-pair sequencesadjacent or in proximity to the target sequence and, depending on thenuclease, can be 5′ or 3′ to the target sequence. Engineering of thePAM-interacting domain of a nucleic acid-guided nuclease may allow foralteration of PAM specificity, improve fidelity, or decrease fidelity.In certain embodiments, the genome editing of a target sequence bothintroduces a desired DNA change to a target sequence, e.g., the genomicDNA of a cell, and removes, mutates, or renders inactive a proto-spacermutation (PAM) region in the target sequence. Rendering the PAM at thetarget sequence inactive precludes additional editing of the cell genomeat that target sequence, e.g., upon subsequent exposure to a nucleicacid-guided nuclease complexed with a synthetic guide nucleic acid inlater rounds of editing. Thus, cells having the desired target sequenceedit and an altered PAM can be selected using a nucleic acid-guidednuclease complexed with a synthetic guide nucleic acid complementary tothe target sequence. Cells that did not undergo the first editing eventwill be cut rendering a double-stranded DNA break, and thus will notcontinue to be viable. The cells containing the desired target sequenceedit and PAM alteration will not be cut, as these edited cells no longercontain the necessary PAM site and will continue to grow and propagate.

The range of target sequences that nucleic acid-guided nucleases canrecognize is constrained by the need for a specific PAM to be locatednear the desired target sequence. As a result, it often can be difficultto target edits with the precision that is necessary for genome editing.It has been found that nucleases can recognize some PAMs very well(e.g., canonical PAMs), and other PAMs less well or poorly (e.g.,non-canonical PAMs). Because certain of the engineered MAD70-seriesnucleases disclosed herein recognize different PAMs, the engineeredMAD70-series nucleases increase the number of target sequences that canbe targeted for editing; that is, engineered MAD70-series nucleasesdecrease the regions of “PAM deserts” in the genome. Thus, theengineered MAD70-series nucleases expand the scope of target sequencesthat may be edited by increasing the number (variety) of PAM sequencesrecognized. Moreover, cocktails of engineered MAD70-series nucleases maybe delivered to cells such that target sequences adjacent to severaldifferent PAMs may be edited in a single editing run.

Another component of the nucleic acid-guided nuclease system is thedonor nucleic acid. In some embodiments, the donor nucleic acid is onthe same polynucleotide (e.g., editing vector or editing cassette) asthe guide nucleic acid and may be (but not necessarily) under thecontrol of the same promoter as the guide nucleic acid (e.g., a singlepromoter driving the transcription of both the guide nucleic acid andthe donor nucleic acid). The donor nucleic acid is designed to serve asa template for homologous recombination with a target sequence nicked orcleaved by the nucleic acid-guided nuclease as a part of thegRNA/nuclease complex. A donor nucleic acid polynucleotide may be of anysuitable length, such as about or more than about 20, 25, 50, 75, 100,150, 200, 500, or 1000 nucleotides in length. In certain preferredaspects, the donor nucleic acid can be provided as an oligonucleotide ofbetween 20-300 nucleotides, more preferably between 50-250 nucleotides.The donor nucleic acid comprises a region that is complementary to aportion of the target sequence (e.g., a homology arm). When optimallyaligned, the donor nucleic acid overlaps with (is complementary to) thetarget sequence by, e.g., about 20, 25, 30, 35, 40, 50, 60, 70, 80, 90or more nucleotides. In many embodiments, the donor nucleic acidcomprises two homology arms (regions complementary to the targetsequence) flanking the mutation or difference between the donor nucleicacid and the target template. The donor nucleic acid comprises at leastone mutation or alteration compared to the target sequence, such as aninsertion, deletion, modification, or any combination thereof comparedto the target sequence.

As mentioned previously, often the donor nucleic acid is provided as anediting cassette, which is inserted into a vector backbone where thevector backbone may comprise a promoter driving transcription of thegRNA and the coding sequence of the gRNA, or the vector backbone maycomprise a promoter driving the transcription of the gRNA but not thegRNA itself. Moreover, there may be more than one, e.g., two, three,four, or more guide nucleic acid/donor nucleic acid cassettes insertedinto an engine vector, where each guide nucleic acid is under thecontrol of separate different promoters, separate like promoters, orwhere all guide nucleic acid/donor nucleic acid pairs are under thecontrol of a single promoter. In some embodiments—such as embodimentswhere cell selection is employed—the promoter driving transcription ofthe gRNA and the donor nucleic acid (or driving more than one gRNA/donornucleic acid pair) is an inducible promoter. Inducible editing isadvantageous in that singulated cells can be grown for several to manycell doublings before editing is initiated, which increases thelikelihood that cells with edits will survive, as the double-strand cutscaused by active editing are largely toxic to the cells. This toxicityresults both in cell death in the edited colonies, as well as a lag ingrowth for the edited cells that do survive but must repair and recoverfollowing editing. However, once the edited cells have a chance torecover, the size of the colonies of the edited cells will eventuallycatch up to the size of the colonies of unedited cells. See, e.g., U.S.Ser. No. 16/399,988, filed 30 Apr. 2019; Ser. No. 16/454,865 filed 26Jun. 2019; and Ser. No. 16/540,606, filed 14 Aug. 2019. Further, a guidenucleic acid may be efficacious directing the edit of more than onedonor nucleic acid in an editing cassette; e.g., if the desired editsare close to one another in a target sequence.

In addition to the donor nucleic acid, an editing cassette may compriseone or more primer sites. The primer sites can be used to amplify theediting cassette by using oligonucleotide primers; for example, if theprimer sites flank one or more of the other components of the editingcassette.

Also, as described above, the donor nucleic acid may comprise—inaddition to the at least one mutation relative to a target sequence—oneor more PAM sequence alterations that mutate, delete or render inactivethe PAM site in the target sequence. The PAM sequence alteration in thetarget sequence renders the PAM site “immune” to the nucleic acid-guidednuclease and protects the target sequence from further editing insubsequent rounds of editing if the same nuclease is used.

In addition, the editing cassette may comprise a barcode. A barcode is aunique DNA sequence that corresponds to the donor DNA sequence such thatthe barcode can identify the edit made to the corresponding targetsequence. The barcode typically comprises four or more nucleotides. Insome embodiments, the editing cassettes comprise a collection of donornucleic acids representing, e.g., gene-wide or genome-wide libraries ofdonor nucleic acids. The library of editing cassettes is cloned intovector backbones where, e.g., each different donor nucleic acid isassociated with a different barcode.

Additionally, in some embodiments, an expression vector or cassetteencoding components of the nucleic acid-guided nuclease system furtherencodes an engineered MAD70-series nuclease comprising one or morenuclear localization sequences (NLSs), such as about or more than about1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. In some embodiments, theengineered nuclease comprises NLSs at or near the amino-terminus, NLSsat or near the carboxy-terminus, or a combination.

The engine and editing vectors comprise control sequences operablylinked to the component sequences to be transcribed. As stated above,the promoters driving transcription of one or more components of theengineered MAD70-series nuclease editing system may be inducible, and aninducible system is likely employed if selection is to be performed. Anumber of gene regulation control systems have been developed for thecontrolled expression of genes in plant, microbe, and animal cells,including mammalian cells, including the pL promoter (induced by heatinactivation of the CI857 repressor), the pBAD promoter (induced by theaddition of arabinose to the cell growth medium), and the rhamnoseinducible promoter (induced by the addition of rhamnose to the cellgrowth medium). Other systems include the tetracycline-controlledtranscriptional activation system (Tet-On/Tet-Off, Clontech, Inc. (PaloAlto, Calif.); Bujard and Gossen, PNAS, 89(12):5547-5551 (1992)), theLac Switch Inducible system (Wyborski et al., Environ Mol Mutagen,28(4):447-58 (1996); DuCoeur et al., Strategies 5(3):70-72 (1992); U.S.Pat. No. 4,833,080), the ecdysone-inducible gene expression system (Noet al., PNAS, 93(8):3346-3351 (1996)), the cumate gene-switch system(Mullick et al., BMC Biotechnology, 6:43 (2006)), and thetamoxifen-inducible gene expression (Zhang et al., Nucleic AcidsResearch, 24:543-548 (1996)) as well as others.

Typically, performing genome editing in live cells entails transformingcells with the components necessary to perform nucleic acid-guidednuclease editing. For example, the cells may be transformedsimultaneously with separate engine and editing vectors; the cells mayalready be expressing the engineered MAD70-series nuclease (e.g., thecells may have already been transformed with an engine vector or thecoding sequence for the engineered MAD70-series nuclease may be stablyintegrated into the cellular genome) such that only the editing vectorneeds to be transformed into the cells; or the cells may be transformedwith a single vector comprising all components required to performnucleic acid-guided nuclease genome editing.

A variety of delivery systems can be used to introduce (e.g., transformor transfect) nucleic acid-guided nuclease editing system componentsinto a host cell. These delivery systems include the use of yeastsystems, lipofection systems, microinjection systems, biolistic systems,virosomes, liposomes, immunoliposomes, polycations, lipid:nucleic acidconjugates, virions, artificial virions, viral vectors, electroporation,cell permeable peptides, nanoparticles, nanowires, exosomes.Alternatively, molecular trojan horse liposomes may be used to delivernucleic acid-guided nuclease components across the blood brain barrier.Of particular interest is the use of electroporation, particularlyflow-through electroporation (either as a stand-alone instrument or as amodule in an automated multi-module system) as described in, e.g., U.S.Pat. No. 10,435,717, issued 8 Oct. 2019; and U.S. Pat. No. 10,443,074,issued 15 Oct. 2019; U.S. Ser. No. 16/550,790, filed 26 Aug. 2019; Ser.No. 10/323,258, issued 18 Jun. 2019; and Ser. No. 10/415,058, issued 17Sep. 2019.

After the cells are transformed with the components necessary to performnucleic acid-guided nuclease editing, the cells are cultured underconditions that promote editing. For example, if constitutive promotersare used to drive transcription of the engineered MAD70-series nucleasesand/or gRNA, the transformed cells need only be cultured in a typicalculture medium under typical conditions (e.g., temperature, CO₂atmosphere, etc.) Alternatively, if editing is inducible—by, e.g.,activating inducible promoters that control transcription of one or moreof the components needed for nucleic acid-guided nuclease editing, suchas, e.g., transcription of the gRNA, donor DNA, nuclease, or, in thecase of bacteria, a recombineering system—the cells are subjected toinducing conditions. The MAD70 nucleases described herein may be used inautomated systems, such as those described in U.S. Pat. No. 10,253,316,issued 9 Apr. 2019; U.S. Pat. No. 10,329,559, issued 25 Jun. 2019; U.S.Pat. No. 10,323,242, issued 18 Jun. 2019; and U.S. Pat. No. 10,421,959,issued 24 Sep. 2019; and U.S. Ser. No. 16/412,195, filed 14 May 2019;Ser. No. 16/423,289, filed 28 May 2019; and Ser. No. 16/571,091, filed14 Sep. 2019.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention, nor are theyintended to represent or imply that the experiments below are all of orthe only experiments performed. It will be appreciated by personsskilled in the art that numerous variations and/or modifications may bemade to the invention as shown in the specific aspects without departingfrom the spirit or scope of the invention as broadly described. Thepresent aspects are, therefore, to be considered in all respects asillustrative and not restrictive.

Example 1: Exemplary Workflow Overview

FIG. 3 shows an exemplary workflow 300 for creating and screeningengineered MAD70-series enzymes. In a first step 301, a wild type MAD7DNA sequence was prepared and cloned to make a template vector forcreation of MAD70-series variants. In another step 303, computerhomology modeling of MAD7 (represented by an amino acid sequence havingthe sequence SEQ ID No. 1) was performed to identify putative regions ofinterest for rationally-designing MAD70 variants with varied PAMpreferences, optimized activity in specific organisms, and alteredfidelity as compared to MAD7. These regions include regions of thenuclease proximal to key regions where it is predicted that the nucleaseinteracts with the PAM, target, or gRNA e.g., see Example 2 below. Onceputative key regions of interest were identified in silico, cassetteswere constructed and cloned into the vector template, then transformedinto cells 305, thereby generating a library of engineered MAD70-seriesvariants. The cells transformed with the engineered MAD70-seriesvariants were arrayed in 96-well plates 307 for storage. At step 309, analiquot of the cells from each well was taken, and the MAD70-seriessequences were amplified from each aliquot. At another step 311, aplasmid expressing a gRNA was constructed, and then combined with theamplified MAD70-series nucleases to perform in vitro transcription andtranslation to make active ribonuclease protein complexes 313. Asynthetic target library was constructed 315, in which to test targetdepletion 317 for each of that MAD70-series variants. After targetdepletion, amplicons were produced for analysis using next-gensequencing 319, and sequencing data analysis was performed 321 todetermine target depletion.

Example 2: Homology Modeling and Positions for Mutation Testing

An in silico homology model of a MAD7 enzyme having the amino acidsequence as represented by SEQ ID No. 1 was made using PDB:5B43structure as a template using SWISS-MODEL(https://swissmodel.expasy.org). Mutation sets were generated based onresidue proximity to putative key regions of where the nuclease ispredicted to interact with the PAM site, target, or gRNA, as well astargeting charged amino acids. The following amino acid residues weretargeted for mutation (the residues are in relation to the MAD7 aminoacid sequence in SEQ ID No. 1): 19, 22, 55, 84, 95, 124, 125, 159, 160,161, 162, 165, 169, 171, 187, 269, 278, 281, 283, 284, 346, 466, 505,511, 517, 528, 529, 530, 531, 532, 533, 534, 535, 536, 539, 582, 584,586, 587, 588, 589, 590, 591, 593, 594, 595, 596, 597, 598, 599, 600,601, 620, 623, 650, 707, 712, 720, 739, 741, 742, 743, 749, 761, 768,785, 786, 822, 830, 833, 842, 853, 878, 881, 912, 920, 924, 925, 932,934, 937, 946, 969, 970, 974, 982, 990, 997, 1019, 1021, 1052, 1054,1109, 1111, 1113, 1173.

Example 3: Vector Cloning, MAD70-Series Variant Library Construction andPCR

The MAD7 coding sequence was cloned into a pUC57 vector with T7-promotersequence attached to the 5′-end of the coding sequence and aT7-terminator sequence attached to the 3′-end of the coding sequence.Next, using a pUC57-MAD7 wildtype vector as a template, a saturatedmutation library for the 96 positions predicted by the modelingdescribed in Example 2 was made substituting the original codon with NNK(IUPAC code for DNA: N=A, T, G, C; K=G, T) randomized codons. Theengineered MAD70-series variants were delivered as a pool of mutantplasmids. 100 ng of a plasmid mixture was transformed into five E.cloni®SUPREME electrocompetent solo cells (Lucigen). After the cells wererecovered in 5 mL of recovery medium at 37° C. for 1 hr in a shakingincubator, 1 mL of 50% glycerol was added and the cells were stored at−80° C. as 100 μL aliquots.

The stored cells were diluted in phosphate buffered saline and spread onLB agar plates with 100 μg/mL of carbenicillin. The cells were thengrown overnight at 37° C. in an incubator. Colonies were picked andinoculated into 1 mL of LB medium (100 μg/mL of carbenicillin) in96-well culture blocks. Cultures were grown overnight in a shakingincubator at 37° C. Next, 1 μL of the cells were diluted into 500 μL ofPCR grade water, and 25 μl aliquots of diluted cultures were boiled for5 min at 95° C. using a thermal cycler. The boiled cells were used toPCR amplify the different engineered MAD70-series variant codingsequences. The rest of the cultures were stored at −80° C. with addedglycerol at 10% v/v concentration.

First, Q5 Hot Start 2× master mix reagent (NEB) was used to amplify theengineered MAD70-series variant sequences using the boiled cells as asource of MAD70-series variant templates. The forward primer5′-TTGGGTAACGCCAGGGTTTT (SEQ ID No. 16) and reverse primer5′-TGTGTGGAATTGTGAGCGGA (SEQ ID No. 17) amplified the sequences flankingthe engineered MAD70-series variant in the pUC57 vector including theT7-promoter and T7-terminator components attached to the MAD7 variantsequence at the 5′- and 3′-end of the engineered MAD70-series variants,respectively. 1 μM primers were used in a 10 μL PCR reaction using 3.3μL boiled cell samples as templates in 96 well PCR plates. The PCRconditions shown in Table 1 were used:

TABLE 1 PCR conditions STEP TEMPERATURE TIME DENATURATION 98° C. 30 SEC30 CYCLES 98° C. 10 SEC 66° C. 30 SEC 72° C. 2.5 MIN FINAL 72° C. 2 MINEXTENSION HOLD 12° C.

Example 4: gRNA Expression Gene Construction in Plasmids and SyntheticTarget Library Construction

Two plasmids were made to produce two different guide RNAs for the invitro depletion assay. A MAD7 gRNA scaffold sequence(5′-GGAATTTCTACTCTTGTAGAT (SEQ ID No. 18)) was placed under the controlof the T7 promoter followed by a guide sequence for synthetic Target 3or Target 7. The sequences of these constructs are shown in FIG. 4.

Two different synthetic target sequences were used to design a syntheticplasmid target library, where the target oligo pools were ordered fromTwist Bioscience (Carlsbad, Calif.) using the following designs: Targetsequence:

Target3: (SEQ ID No. 19) 5'-CCAGTCAGTAATGTTACTGG, and Target7:(SEQ ID No. 20) 5'-AGCAGGACACTCCTGCCCCA.

TABLE 2 Target Library Design: NUMBER OF TARGET PAMs FOR VARIANT/LIBRARY PAM 5′ 3′ UMI ANALYSIS DESIGN PAM PANEL TNNN N 64 1 SPECIFICITYTTTV N 3 12 PANEL

The PAM panel library was designed by adding TNNN randomized sequencesas the 5′-end PAM for each target, then by adding a single bp N at theend of the target to be used as the unique molecular identifier in thesequencing analysis. The specificity panel was designed by introducing 2bp tandem mismatches in the following positions in each target: 1st,3rd, 7th, 8th, 9th, 11th, 13th, 14th, 15th, 17th, 18th, and 19th bp.Each target with 2 bp mismatches was used to add 5′-end TTTV PAM (IUPACnomenclature: V=A, G, or C) and 3′-end 1 bp N as the UMI (uniquemolecule identifier) for sequencing analysis. The target library wascloned into a pUC19 backbone and prepared using the Midi-plus™ plasmidpreparation kit (Qiagen). The target library pool was prepared at 10ng/μL final concentration.

Example 5: In Vitro Transcription and Translation for Production ofMAD70-Series Nucleases and gRNAs in a Single Well

A PURExpress® In Vitro Protein Synthesis Kit (NEB) was used to produceengineered MAD70-series variant proteins from the PCR-amplifiedMAD70-series variant library, and also to produce gRNAs for synthetictarget Library of Target3 and Target7. In each well in a 96-well plate,the reagents in Table 3 were mixed to start the production of MAD7variants and gRNA:

TABLE 3 Reagents REAGENTS VOLUME (μl) 1 SolA (NEB kit) 3.3 2 SolB (NEBkit) 2.5 3 gRNA mix (4 ng/μl stock) 0.8 4 Murine RNase inhibitor (NEB)0.2 5 Water 0.5 6 PCR amplified T7 MAD70-series variants 1.0

A master mix with all reagents was mixed on ice with the exception ofthe PCR-amplified T7-MAD70-series variants to cover enough 96-wellplates for the assay. After 7.3 μL of the master mix was distributed ineach well in 96 well plates, 1 μL of the PCR amplified MAD70-seriesvariants under the control of T7 promoter was added. The 96-well plateswere sealed and incubated for 4 hrs at 37° C. in a thermal cycler. Theplates were kept at room temperature until the target pool was added toperform the target depletion reaction.

Example 6: Performing Target Depletion, PCR and NGS

After 4 hours incubation to allow production of the engineeredMAD70-series variants and gRNAs, 4 μL of the target library pool (10ng/μL) was added to the in vitro transcription/translation reactionmixture. After the target library was added, reaction mixtures wereincubated overnight at 37° C. The target depletion reaction mixtureswere diluted into PCR-grade water that contains RNAse A and then boiledfor 5 min at 95° C. The mixtures were then amplified and sequenced. ThePCR conditions in Table 4 below were used:

TABLE 4 PCR Conditions STEP TEMPERATURE TIME DENATURATION 98° C. 30 SEC 6 CYCLES 98° C. 10 SEC 61° C. 30 SEC 72° C. 10 SEC 22 CYCLES 98° C. 10SEC 72° C. 10 SEC FINAL 72° C. 2 MINUTES EXTENSION HOLD 12° C.

Example 7: Data Analysis

Table 5 is a table of amino acid substitutions made to the MAD7 nucleaseamino acid sequence (SEQ ID No. 1) that result in MAD70-series variantnucleases with different PAM recognition sites as compared to the nativeMAD7 nuclease.

TABLE 5 MAD70-series Variants - Altered PAM Preference Mutation NewPAMs, cut WT Residue Detected detected SEQ ID No. K535L L TGTN, TTCN SEQID No. 2 K535S S TGTN, TTCN SEQ ID No. 11 K535C C TGTN, TTCN SEQ ID No.12 K535R R TGTN, TTCN SEQ ID No. 13 K535N N TGTN, TTCN SEQ ID No. 14K535G G TCTN as primary SEQ ID No. 3

FIG. 1A is a heatmap for the MAD70-series variant nucleases withdifferent PAM recognition sites. The K535R mutation disrupts the abilityof the enzyme to recognize TCTN PAMs and enhances the ability of theenzyme to recognize PAMs containing a purine at the second position(TATN/TGTN). The K594 mutation ablates the recognition of the preferredTTTN PAMs while enhancing TCGN recognition.

Table 6 is a table of amino acid substitutions made to the MAD7 nucleaseamino acid sequence (SEQ ID No. 1) resulting in MAD70-series variantnucleases with varied targeting fidelity as compared to the MAD7reference nuclease.

TABLE 6 MAD70-series Variants - Varied Target Fidelity Mutation Pos_9score for HF- WT Residue Detected MAD7 (wt > 0.3) SEQ ID No. R920G G 0.0SEQ ID No. 4 R924I I 0.04 SEQ ID No. 5 K511L L 0.03 SEQ ID No. 6 H283T T0.01 SEQ ID No. 7 R187K K 0.0 SEQ ID No. 8 N589G G 0.0 SEQ ID No. 9K281A A 0.04 SEQ ID No. 10 K281V V 0.01 SEQ ID No. 15

FIG. 1B is the heatmap for the MAD70-series variant nucleases withvaried fidelity as compared with wild-type MAD7 (SEQ ID No. 1). Thebottom figure shows the PAM depletion panel for the same enzyme from theabove figure. R187K and N589G showed better pos9 specificity but notefrom the bottom figure these MAD70-series nucleases showed reducedactivity across all PAMs. As can be seen many of these mutationseliminate activity of the enzyme for targets that contain programmed 2bp mismatches at the +9, +14, +15, and +17 positions relative to the PAMsequence indicating an improved targeting fidelity.

FIG. 2A is a PAM depletion vs specificity plot of the native MAD7sequence (SEQ ID No. 1) sampled across multiple plates in a HT-screen.The PAM specificity is represented as the sum of the depletion scoresobserved for all PAMs tested (D_(PAM)) as calculated by Eqn 1:

PAM_(score)=ΣD_(PAM)  eqn.1:

The relative nuclease specificity is calculated as the pos9_score asshown in eqn 2.

$\begin{matrix}{{pos}_{9_{score}} = {\frac{D_{9}}{D_{wt}}:}} & {{eqn}.\mspace{14mu} 2}\end{matrix}$

Where D₉ is the sum of the depletion scores for DNA target sequencescontaining a 2 bp mismatch at the PAM +9 position and D_(wt) is the sumof the depletion scores for DNA targets with perfect complementarity tothe gRNAs used in this assay. This scoring methodology was chosenempirically based on the sensitivity of the targeting specificity tomutations in this register of the RNA-DNA interaction. Each pointcorresponds to an independent measurement from control digestionexperiments run with the MAD7 nuclease (SEQ ID NO. 1). FIG. 2B is theplot for the screened 1108 single amino acid variants tested. Points inthe lower two quadrants represent loss of function mutations whichoccurred in 433/1020 (43%) of the screened space. Data points in theupper left portion of the graph (>10 sum of Pam_depletion, <0.3pos_9_depletion/wt_depletion) represent variants that with high activityas judged by their summed PAM activity score and high altered RNA-guidedenzyme fidelity relative to the wild-type MAD7 enzyme sequence (FIG.2A).

Example 8: Combinatorial Mutation Library Construction, Screening andData Analysis

Based on the results observed in Example 7, an additional mutant librarywas designed and screened for changes in PAM preference. The library wascomposed of mutations at both positions K535 and K594 (the residues arein relation to the MAD7 amino acid sequence in SEQ ID No. 1)substituting the original codon with NNK (IUPAC code for DNA: N=A, T, G,C; K=G, T) randomized codons. The library was constructed using a Q5Site Directed Mutagenesis Kit (NEB) using manufacturers protocols withmutagenic forward 5′-TTCTNNKAACGCTATCATACTGATGC (SEQ ID No. 25) andreverse 5′TACTCMNNGGACTTTGACCAACCGTC (SEQ ID No. 26) primers. The PCRreaction mix was transformed into 5-alpha chemically competent cells(NEB) and plated on LB agar plates with 100 μg/mL of carbenicillin.Colonies were picked and inoculated into 1 mL of LB medium (100 m/mL ofcarbenicillin) in 96-well culture blocks and grown overnight in ashaking incubator at 37° C. Sample processing and screening wasperformed as described in examples 3, 4, 5 and 6.

FIG. 5 is a heatmap for a MAD70-series nuclease with novel PAMrecognition sites identified from this library screening (SEQ ID No.67). This mutant contains the combination of mutations K535R and N539S(SEQ ID No. 67) and results in more robust activity on PAMs with an Anucleotide at the second position of the NNNN PAM space, in particularTAAN, compared to the K535R mutation alone.

Example 9: Revised Target Library

A revised PAM panel library was designed by adding NNNN randomizedsequences as the 5′-end PAM for each target, in order to evaluateactivity on all 256 PAM sequences in the NNNN PAM space. Oligo poolswere ordered from Twist Bioscience (Carlsbad, Calif.) using thefollowing designs: Target3: 5′-CCAGTCAGTAATGTTACTGG (SEQ ID No. 27), andTarget7: 5′-AGCAGGACACTCCTGCCCCA (SEQ ID No. 28). The target library wascloned into a pUC19 backbone and prepared using the Midi-plus™ plasmidpreparation kit (Qiagen). The target library pool was prepared at 10ng/μL final concentration.

TABLE 7 Revised Library Number of Target PAMs for Variant/ Library PAM5′ 3′UMI analysis Design PAM panel NNNN none 256 1 (2 targets)

Example 10: Mutagenic Library Construction, Screening and Data AnalysisUsing K535R/N539S Backbone

In order to further alter the PAM preference, a library of single aminoacid mutations was generated using the K535R/N539S mutant (SEQ ID No.67) described in Example 8. Mutation sets were generated based onresidue proximity to putative key regions of where the nuclease ispredicted to interact with the PAM site, target, or gRNA, as well astargeting charged amino acids. The following amino acid residues weretargeted for mutation (the residues are in relation to the MAD7 aminoacid sequence in SEQ ID No. 1): 529, 530, 531, 532, 534, 536, 537, 538,540, 541, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593,594, 595, 599, 601, 650, 739, 740, 741, 742, 743. At each position, theoriginal codon was substituted with NNK (IUPAC code for DNA: N=A, T, G,C; K=G, T) randomized codons. This library was screened for altered PAMpreference as described in examples 3, 4, 5 and 6, using the targetoligonucleotide library described in Example 9.

FIG. 6 represents activity heatmaps for wild-type MAD7 (SEQ ID No. 1)(FIG. 6A), the K535R/N539S mutant (SEQ ID No. 67) (FIG. 6B) used as theparent for this library, along with an additional MAD70-series nucleasewith novel PAM recognition sites identified from this library (SEQ IDNo. 68) (FIG. 6C). Data analysis was performed as described in Example7, with heatmaps now representing activity on all 64 combinations ofnucleotides in the NNNN PAM space. The new MAD70-series nuclease mutant(SEQ ID No. 68) contains the combination of mutationsK535R/N539S/K594L/E730Q in relation to the wild-type MAD7 amino acidsequence in SEQ ID No. 1. It has novel activity on PAMs with a Cnucleotide at the third position of the NNNN PAM space.

Example 11: Activity of MAD70-Series PAM Mutants in Escherichia coliCells

In order to confirm activity of the MAD70-series mutants for genomeediting systems in cells, activity was confirmed using a phenotypicediting assay in E. coli. MAD70-series mutants were cloned into a EE0026vector backbone. MAD70-series variants were amplified using reverse (5′GATGATTTCTCTAGAGGTACTTAGAGATAGCGCTTATTCTGGATAAAGTC) (SEQ ID No. 29) andforward (5′ CGATTCCGGAAAGGAGATATCTCATGAACAACGGCACAAATAATTTTCAG AA) (SEQID No. 30) primers and cloned into the linearized EE0026 Engine vectorusing the NEBuilder HF DNA assembly kit.

Editing cassettes were designed to introduce stop codons to disrupt thesynthesis of full-length LacZ in E. coli as a result of editing. Eachcassette was composed of a 20 base pair spacer to precisely target aregion of lacZ gene in the E. coli genome adjacent to the indicated PAMsequence in the genome, and a 200 bp repair template for homologousrecombination. DNA sequences and corresponding PAM targets for eachcassette are provided in Table 8. Each cassette is cloned into thecommon cassette vector backbone p346BB (SEQ ID No. 87) using theNEBuilder HF DNA assembly kit. E. coli K-12 str MG1655 grown to mid-logphase in LB was made electrocompetent by washing three times with icecold 10% glycerol. Engine vectors were transformed by electroporation,recovered in SOC for 1 hr at 30° C., then grown overnight on LB agarwith Chloramphenicol (25 ug/mL) medium at 30° C. Overnight grown cellswith MAD70-series variant engine vectors were grown to mid log phase inLB Chloramphenicol (25 ug/mL) and made competent with LB brothcontaining 10% (wt/vol) polyethylene glycol, 5% (vol/vol) dimethylsulfoxide, and 50 mM Mg2+ at pH 6.5.

TABLE 8 Sequences of Editing cassettes and corresponding PAM targetsTarget SEQ ID Cassette name Insert sequence gene PAM No.lacZ_127_TTTC_stop GTGTGTGATACGAAACGAAGCATTGGAGGCATTG lacZ TTTC 31GAATTTCTACTCTTGTAGATACCCTGCCATAAAGA AACTGTCCATGTTGCCACTCGCTTTAATGATGATTTCAGCCGCGCTGTACTGGAGGCTGAAGTTCAGAT GTGCGGCGAGTTGCGTGACTACCTACGGGTAACATAATGATTATGGTAATGAGAGACCCAGGTCGCCA GCGGCACCGCGCCTTTCGGCGGTGAAATTATCGATGAGCGTGGTGGTTATGCCGATCGCGTCACACTA ATCCCAGAAAAGACCCGTCCGlacZ_245_TTTC_stop GTGTGTGATACGAAACGAAGCATTGGAGGCATTG lacZ TTTC 32GAATTTCTACTCTTGTAGATCATGTTGCCACTCGC TTTAATCACCCTGCCATAAAGAAACTGTTACCCGTAGGTAGTCACGCAACTCGCCGCACATCTGAACTT CAGCCTCCAGTACAGCGCGGCTGAAATCATCATTTCATTAAGTGGCTCATTAGAGATAGCTGATTTGTGT AGTCGGTTTATGCAGCAACGAGACGTCACGGAAAATGCCGCTCATCCGCCACATATCCTGATCTTCATC CCAGAAAAGACCCGTCCGlacZ_256_TTTG_stop GTGTGTGATACGAAACGAAGCATTGGAGGCATTG lacZ TTTG 33GAATTTCTACTCTTGTAGATTGTAGTCGGTTTATG CAGCAACCGCCTCGCGGTGATGGTGCTGCGCTGGAGTGACGGCAGTTATCTGGAAGATCAGGATATGT GGCGGATGAGCGGCATTTTCCGTGACGTCTCGTTGTAATGAAAACCGTAATGACAGATTAGCGATTTCC ATGTTGCCACTCGCTTTAATGATGATTTCAGCCGCGCTGTACTGGAGGCTGAAGTTCAGATGTGCGGCA TCCCAGAAAAGACCCGTCCGlacZ_419_TTTG_stop GTGTGTGATACGAAACGAAGCATTGGAGGCATTG lacZ TTTG 34GAATTTCTACTCTTGTAGATCCGTCTGAATTTGAC CTGAGCTCATCCGCCACATATCCTGATCTTCCAGATAACTGCCGTCACTCCAGCGCAGCACCATCACCG CGAGGCGGTTTTCTCCGGCGCGTAAAAATGCGCTTCATTAAAATTCTCATTACAGACCACTGTCCTGGCC GTAACCGACCCAGCGCCCGTTGCACCACAGATGAAACGCCGAGTTAACGCCATCAAAAATAATTCGAT CCCAGAAAAGACCCGTCCGlacZ_314_TATG_stop GTGTGTGATACGAAACGAAGCATTGGAGGCATTG lacZ TATG 35GAATTTCTACTCTTGTAGATTGGCGGATGAGCGGC ATTTTTCCAGTACAGCGCGGCTGAAATCATCATTAAAGCGAGTGGCAACATGGAAATCGCTGATTTGTG TAGTCGGTTTATGCAGCAACGAGACGTCACGGAATCATTAGCTCATTCATTACACATTCTGATCTTCCA GATAACTGCCGTCACTCCAGCGCAGCACCATCACCGCGAGGCGGTTTTCTCCGGCGCGTAAAAATGCA TCCCAGAAAAGACCCGTCCGlacZ_920_TATG_stop GTGTGTGATACGAAACGAAGCATTGGAGGCATTG lacZ TATG 36GAATTTCTACTCTTGTAGATACCATGATTACGGAT TCACTCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGG TTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTCATTACGTAATTCATTACACTCATGTTTCCTGT GTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGA TCCCAGAAAAGACCCGTCCGlacZ_1712_AACG_stop GTGTGTGATACGAAACGAAGCATTGGAGGCATTG lacZ AACG 37GAATTTCTACTCTTGTAGATCCATCAAAAATAATT CGCGTATTACGGTCAATCCGCCGTTTGTTCCCACGGAGAATCCGACGGGTTGTTACTCGCTCACATTTAA TGTTGATGAAAGCTGGCTACAGGAAGGCCAGACGTAATGAATTTTTTAATGAGTCAATTCGGCGTTTCA TCTGTGGTGCAACGGGCGCTGGGTCGGTTACGGCCAGGACAGTCGTTTGCCGTCTGAATTTGACCTATC CCAGAAAAGACCCGTCCGlacZ_466_AACG_stop GTGTGTGATACGAAACGAAGCATTGGAGGCATTG lacZ AACG 38GAATTTCTACTCTTGTAGATGGGATACTGACGAAA CGCCTAATGGCTTTCGCTACCTGGAGAGACGCGCCCGCTGATCCTTTGCGAATACGCCCACGCGATGG GTAACAGTCTTGGCGGTTTCGCTAAATACTGGCAGTAATGACGTCAGTAATGACGACTTCAGGGCGGCT TCGTCTGGGACTGGGTGGATCAGTCGCTGATTAAATATGATGAAAACGGCAACCCGTGGTCGGCTTAC ATCCCAGAAAAGACCCGTCCG10 ng of editing cassette plasmid was added to 20 uL of chemicallycompetent E. coli strain with an engine vector on ice. After 30 min, 250uL of SOC was added and the cultures were incubated in a shakingincubator for 1 hr at 30° C. 30 uL of the resulting cultures wereinoculated to 350 uL of LB Carbenicillin (100 ug/mL)/Chloramphenicol (25ug/mL) and grown overnight in a shaking incubator at 30° C. 4 uL of theovernight cultures were inoculated to fresh 320 uL LB/Carbenicillin (100ug/mL)/Chloramphenicol (25 ug/mL)/Arabinose (1% w/v) medium andincubated for 3 hrs in a shaking incubator at 30° C. Cultures were movedto a 42° C. shaking incubator to induce the production of RNP complex.After 5 hrs of induction at 42° C., cultures were moved back to the 30°C. shaking incubator and grown overnight. Overnight grown edited strainswere spotted on a MacConkey Agar Plates (Teknova) and grown overnight at37° C. without any antibiotics. Cultures with native LacZ can fermentthe lactose in the medium, produces acid that lowers the pH that makesthe red color in the colony. Cultures with edited disrupted LacZ can'tferment the lactose and the colonies grow colorless. A summary ofediting phenotypes observed for MAD70-series PAM mutants is shown inFIG. 7. Darker spots indicate intact lacZ and lighter spots are cellswith lacZ that are edited and thus are non-functional, indicating geneediting activity on the PAMs listed at top.

Example 12: Phenotypic Assay to Measure Genome Editing for MAD7-DerivedMutants in Saccharomyces cerevisiae Cells

To assess the genome editing activity of RNA-guided nucleases in S.cerevisiae, a two micron plasmid was constructed for the sequentialintroduction of DNA containing an editing cassette with SNR52promoter-driven crRNA and a CYC1 promoter-driven nuclease protein (seeFIG. 8). The editing cassette comprises the crRNA to guide the nucleaseto cut at a specific DNA sequence, a short linker, and a repair templatecontaining the mutation of interest flanked by regions of homology tothe genome. The screening plasmid (FIG. 8) was linearized by the StuIrestriction endonuclease, and the editing cassette was introduceddownstream of the SNR52p promoter by isothermal assembly. The editingcassettes inserted into the StuI-linearized plasmid for the introductionof a premature stop codon into the can1 gene, organized by the PAM ofthe corresponding spacer, are shown in Table 9. The nuclease proteinswere amplified by polymerase chain reaction with oligonucleotide primersto introduce an SV40 nuclear localization sequence at the N-terminusconsisting of the DNA sequence “ATGGCACCCAAGAAGAAGAGGAAGGTGTTA” (SEQ IDNo. 39) corresponding to a protein sequence of “MAPKKKRKVL (SEQ ID No.40).” The resulting amplified DNA fragment (400 ng, purified) was thenco-transformed along with a PsiI-linearized screening plasmid (250 ng)that already contained an editing cassette to assemble the completeediting plasmid by in vivo gap repair. Cells containing a repairedplasmid were selected for in yeast peptone-dextrose (YPD) containing 200mg/L Geneticin for 3 days at 30 degrees C. in a humidified shakingincubator. The resulting saturated culture was diluted 1:80 intosynthetic complete yeast media lacking arginine and containing 50 mg/Lof canavanine and grown overnight at 30 degrees C. in a humidifiedshaking incubator. Because knockout of the Can1 protein allows yeast togrow in the presence of the otherwise toxic analog canavanine, therelative OD600 of the overnight cultures is proportional to the rate ofgenome mutation induced by the transformed nuclease protein. TheMAD70-series variants described in Examples 7 and 8 with altered PAMpreference were evaluated in the assay system using the editingcassettes shown in Table 9, targeting various PAMs. The results of thisanalysis are shown in FIG. 9, where the mutant containing mutationsK535R, K539S in reference to the wild-type MAD7 sequence showssubstantially higher editing activity on TATV PAMs.

TABLE 9 Editing cassettes targeting yeast can1 gene tointroduce loss of function mutations SEQ ID Cassette name PAMEditing Cassette Sequence No. Can1_S30stop TTTAGGCCCCAAATTCTAATTTCTACTGTTGTAGATAC 41 GACGTTGAAGCTTCACAATTTTTACGCCGACATAGAGGAGAAGCATATGTACAATGAGCCGGTCAC AACCCTCGAGACACGACGTTGAAGCTTAACAAACACACCACAGACGTGGGTCAATACCATTGAAAG ATGAGAAAAGTAACAATATACGCGCTCCTGCCCCan1_K42stop TTTA GGCCCCAAATTCTAATTTCTACTGTTGTAGATCT 42TTTCTCATCTTTCAATGGTTTTTGTATCCTCGCCA TTTACTCTCGTCGGGAAAGAGCGCAATGGATACAATTCCCCACTTTTCTCATCTTACAATGGTATTG ACCCACGTCTGTGGTGTGTTTGTGAAGCTTCAACGTCGTCAATATACGCGCTCCTGCCC Can1 _N60stop TTTCGGCCCCAAATTCTAATTTCTACTGTTGTAGATCC 43 GACGAGAGTAAATGGCGATTTTTTCAATACCATTGAAAGATGAGAAAAGTAAAGAATTGTATCCAT TGCGCTCGTTCCCGACGAGAGTATAAGGCGAGGATACGTTCTCTATGGAGGATGGCATAGGTGATG AAGATGAAGGAGAAGCAATATACGCGCTCCTGC CCCan1_T115stop TTTA GGCCCCAAATTCTAATTTCTACTGTTGTAGATTC 44CACACCTCTGACCAACGCTTTTTATTGGTATGAT TGCCCTTGGTGGTACTATTGGTACAGGTCTTTTCATTGGATTATCCACACCTCTGTAAAACGCCGGC CCAGTGGGCGCTCTTATATCATATTTATTTATGGGTTCTTTGGCATCAATATACGCGCTCCTGCCC Can1_Q158stop TTTCGGCCCCAAATTCTAATTTCTACTGTTGTAGATAC 45 AGTTTTCTCACAAAGATTTTTTTTCTGTCACGCAGTCCTTGGGTGAAATGGCTACATTCATCCCTGTT ACATCCTCGTTCACAGTTTTCTCATAAAGATTCCTTTCTCCAGCATTTGGTGCGGCCAATGGTTACAT GTATTGGTTTTCAATATACGCGCTCCTGCCCCan1_I214stop TTTG GGCCCCAAATTCTAATTTCTACTGTTGTAGATGG 46TAATTATCACAATAATGATTTTTCATTCAATTTT GGACGTACAAAGTTCCACTGGCGGCATGGATTAGTATTTGGAAGGTAATTATCACATAAATGAACT TGTTCCCTGTCAAATATTACGGTGAATTCGAGTTCTGGGTCGCCAATATACGCGCTCCTGCCC Can1_G72stop TCTAGGCCCCAAATTCTAATTTCTACTGTTGTAGATTG 47 GAGGATGGCATAGGTGATTTTTTAATTGTATCCATTGCGCTCTTTCCCGACGAGAGTAAATGGCGAG GATACGTTCTCCATGGAGGATGGCATATAAGATGAAGATGAAGGAGAAGTACAGAACGCTGAAGT GAAGAGAGAGCTTAACAATATACGCGCTCCTGC CCCan1_Q80stop TCTC GGCCCCAAATTCTAATTTCTACTGTTGTAGATTT 48CACTTCAGCGTTCTGTACTTTTTCCAATAGTACC ACCAAGGGCAATCATACCAATATGTCTTTGCTTAAGCTCCCCCTTCACTTCAGCGTTTTATACTTCT CCTTCATCTTCATCACCTATGCCATCCTCCATAGAGAACGTATCAATATACGCGCTCCTGCCC Canl_E142stop TGTCGGCCCCAAATTCTAATTTCTACTGTTGTAGATAC 49 GCAGTCCTTGGGTGAAATTTTTTCCAGTGGGCGCTCTTATATCATATTTATTTATGGGTTCTTTGGCAT ATTCGGTCACGCAGTCCTTGGGTTAAATGGCTACATTCATCCCTGTTACATCCTCTTTCACAGTTTTC TCACAAAGATCAATATACGCGCTCCTGCCCCan1_S152stop TGTG GGCCCCAAATTCTAATTTCTACTGTTGTAGATAG 50AAAACTGTGAAAGAGGATTTTTTAACCAATACA TGTAACCATTGGCCGCACCAAATGCTGGAGAAAGGAATCTCCCTGAGAAAACTGTGAATTAGGATG TAACAGGGATGAATGTAGCCATTTCACCCAAGGACTGCGTGACAGCAATATACGCGCTCCTGCCC Can1_V20stop TATGGGCCCCAAATTCTAATTTCTACTGTTGTAGATTA 51 CAATGAGCCGGTCACAACTTTTTGGCATAGCAATGACAAATTCAAAAGAAGACGCCGACATAGAG GAGAAGCACGGGTACAATGAGCCGTAAACAACCCTCTTTCACGACGTTGAAGCTTCACAAACACA CCACAGACGTGGGTCAACAATATACGCGCTCCT GCCCCan1_N116stop TATC GGCCCCAAATTCTAATTTCTACTGTTGTAGATCA 52CACCTCTGACCAACGCCGTTTTTGTATGATTGCC CTTGGTGGTACTATTGGTACAGGTCTTTTCATTGGTTTAAGTACACCTCTGACCTAAGCCGGCCCAG TGGGCGCTCTTATATCATATTTATTTATGGGTTCTTTGGCATATTCCAATATACGCGCTCCTGCCC

Example 13: Testing of MAD7 Variant Proteins for Enhanced Genome Editingin S. cerevisiae

To screen libraries of MAD7 enzyme variants with one or more mutationsfor increased genome editing activity in S. cerevisiae, six differentediting cassettes (all targeting the TTTV PAM class) (first six entriesin Table 9 (SEQ ID Nos. 41-46)) were inserted into the StuI-linearizedtwo micron screening plasmid (again see FIG. 8) as described in Example12. MAD7 protein variant coding sequences as described in Example 2 wereamplified by polymerase chain reaction with oligonucleotide primers tointroduce an SV40 nuclear localization sequence at the N-terminusconsisting of the DNA sequence “ATGGCACCCAAGAAGAAGAGGAAGGTGTTA” (SEQ IDNo. 39) corresponding to a protein sequence of “MAPKKKRKVL (SEQ ID No.40).” The resulting amplified DNA fragment (5 uL of crude PCR mixture)was then co-transformed along with a PsiI-linearized screening plasmid(150 ng total, a pool of all 6 editing cassettes) that already containsan editing cassette to assemble the complete editing plasmid by in vivogap repair. Cells containing a repaired plasmid were selected for inyeast peptone-dextrose (YPD) containing 200 mg/L Geneticin for 3 days at30° C. in a humidified shaking incubator. The resulting saturatedculture was diluted into synthetic complete yeast media lacking arginineand containing 50 mg/L of canavanine and grown overnight at 30° C. in ahumidified shaking incubator. Because knockout of the Can1 proteinallows yeast to grow in the presence of the otherwise toxic analogcanavanine, the relative OD600 of the overnight cultures is proportionalto the rate of genome mutation induced by the transformed nucleaseprotein. The relative genome editing activity levels of each variant areplotted in FIG. 10. Rescreening of the variants in quadruplicate in theoriginal assay confirmed the enhanced genome editing activity of severalMAD70-series variants, as shown in FIG. 11. Sequences are provided inSEQ ID Nos. 69 (K95L), 70 (V201I/K278T), 71 (K511D), 72 (N589H), 73(L597V), 74 (K712V), 75 (E743I), 76 (K786S), 77 (K853R), and 78(R1113F).

Example 14: Generation of Combinatorial MAD7 Variant Libraries andScreening for Enhanced Editing in S. cerevisiae

Based on the identified single mutations that enhance the genome editingactivity of MAD7 in S. cerevisiae, combinatorial libraries wereprepared. The N589H MAD70-series variant sequence (SEQ ID No. 72) wasused as a backbone and 4 to 5 additional mutations were introduced usingoligonucleotide primers and the Quick-Change Lightning Multi-SiteMutagenesis kit (Agilent) according to manufacturer instructions. Thesevariants were screened for genome editing activity in S. cerevisiae asdescribed in Example 12 as depicted in FIG. 12. The variants that showedenhanced activity in the primary screen were rescreened in quadruplicateand the results of the secondary screening are depicted in FIG. 13.Sequences are provided in SEQ ID Nos. 79(S124T/K511I/N589H/K712V/K853R/H946W), 80(S124T/K511I/N589H/K786S/H946K/R113F), 81(K511H/N589H/K853R/K1021L/D118E/DE11833), 82 (K95L/S124T/K511I/N589H),83 (S124T/K511I/N589H/K7211/K786S/K1021V), 84(5124T/K511H/N589H/K853R/H946K/K1054Y), 85(K95T/S124T/N589H/K853R/K1052Q), and 86(S124T/K511T/N589H/K712L/H946T/K1052Q/K1054N).

Example 15: Activity of MAD70-Series PAM Mutants in Mammalian Cells

Wild-type MAD7 and MAD70-series variants with altered PAM preferencewere cloned downstream of a CAG promoter for strong expression inmammalian cells. The vector sequence used for expression is provided inSEQ ID No. 89. Guide RNAs (gRNAs) targeting various PAMs were cloneddownstream of a U6 promoter in the backbone vector sequence provided inSEQ ID No. 90. Transfections in HEK293T cells were performed using 100ng of total DNA (gRNA/MAD70-series variant plasmid) and Lipofectamine3000 transfection reagent. The transfection mix was added to cells thathad been cultured in 96 well plates 24 hrs prior to transfection. Tomeasure indels, T7E1 assay was performed. Cells were lysed by theaddition of a buffer containing proteinase K and incubation at 56° C.for 30 minutes. Proteinase K was inactivated by heating the reaction to95° C. for 10 minutes. Following lysis, 10 uL PCR reactions wereperformed using genomic template from lysed cells and 2×Q5 PCR mastermix(NEB) to amplify amplicons containing the target sites that were edited.Following PCR, the PCR fragments were heated to 95° C. C for 5 minutesand slowly cooled to room temperature. Then, T7 endonuclease I (NEB) wasadded to the PCR reaction and incubated for 1 hour at 37° C. Thereaction was then resolved on 2.5% agarose gel and imaged using GelDoc(BioRad). The band intensities on the gel were quantified to calculateindels introduced by MAD7. The results are shown in FIG. 14. TheMAD70-series mutant containing mutations K535R/N539S (SEQ ID No. 67) inreference to the wild-type MAD7 sequence shows substantially higherediting activity on TATC PAM while the K535R/N539S/K594L/E730Q (SEQ IDNo. 68) mutant in relation to wild-type MAD7 shows higher editing onATCC and TTCC PAMs.

6Table 10: Sequences of Spacers and the PAM Sequences that were Targetedin the PPIB Locus

Target SEQ ID # PAM Spacer Sequence No. 1 CTTC cctcccctagcaacgcccctt 532 CATA ggatttttaccgtcaccaaaa 54 3 AATA tggctctattctctctcccat 55 4 ATCGgctgaactctgcaggtcagtt 56 5 ATCC tcaggttagcttcttgtacct 57 6 AATCagattcagaaccacttctcta 58 7 TATC ctgtagtccaaggagggtata 59 8 TATAgataagcatgttttccaagaa 60 9 AACG cccctttaaagaagctaagtt 61 10 AACCacttctctaaaaatatggctc 62 11 TTTT tcagattcagaaccacttctc 63 12 TTTTtatggctctattctctctccc 64 13 ATTC tctctcccatcctcaggttag 65 14 TTCCtcaggtgtattttgacctacg 66

While this invention is satisfied by embodiments in many differentforms, as described in detail in connection with preferred embodimentsof the invention, it is understood that the present disclosure is to beconsidered as exemplary of the principles of the invention and is notintended to limit the invention to the specific embodiments illustratedand described herein. Numerous variations may be made by persons skilledin the art without departure from the spirit of the invention. The scopeof the invention will be measured by the appended claims and theirequivalents. The abstract and the title are not to be construed aslimiting the scope of the present invention, as their purpose is toenable the appropriate authorities, as well as the general public, toquickly determine the general nature of the invention. In the claimsthat follow, unless the term “means” is used, none of the features orelements recited therein should be construed as means-plus-functionlimitations pursuant to 35 U.S.C. § 112, ¶6.

We claim:
 1. An engineered nucleic acid-guided nuclease with a loweredcutting activity relative to the nucleic acid-guided nuclease having thesequence of SEQ ID No. 1, wherein the engineered nucleic acid-guidednuclease has a sequence comprising any of SEQ ID Nos. 8, 9, 10 or
 15. 2.The engineered nucleic acid-guided nuclease of claim 1 comprising SEQ IDNo.
 8. 3. The engineered nucleic acid-guided nuclease of claim 1comprising SEQ ID No.
 9. 4. The engineered nucleic acid-guided nucleaseof claim 1 comprising SEQ ID No.
 10. 5. The engineered nucleicacid-guided nuclease of claim 1 comprising SEQ ID No.
 15. 6. An enzymecocktail comprising an engineered nucleic acid-guided nuclease ofclaim
 1. 7. An enzyme cocktail comprising an engineered nucleicacid-guided nuclease of claim 1 and SEQ ID No.
 2. 8. An enzyme cocktailcomprising an engineered nucleic acid-guided nuclease of claim 1 and SEQID No.
 3. 9. An enzyme cocktail comprising an engineered nucleicacid-guided nuclease of claim 1 and SEQ ID No.
 11. 10. An enzymecocktail comprising an engineered nucleic acid-guided nuclease of claim1 and SEQ ID No.
 12. 11. An enzyme cocktail comprising an engineerednucleic acid-guided nuclease of claim 1 and SEQ ID No.
 13. 12. An enzymecocktail comprising an engineered nucleic acid-guided nuclease of claim1 and SEQ ID No.
 14. 13. An enzyme cocktail comprising an engineerednucleic acid-guided nuclease of claim 1 and SEQ ID No.
 67. 14. An enzymecocktail comprising an engineered nucleic acid-guided nuclease of claim1 and SEQ ID No.
 68. 15. An enzyme cocktail comprising at least two ofthe engineered nucleic acid-guided nucleases of claim
 1. 16. An enzymecocktail comprising at least three of the engineered nucleic acid-guidednucleases of claim
 1. 17. An enzyme cocktail comprising all of theengineered nucleic acid-guided nucleases of claim 1.